Detectors

By far the majority of GC amino acid analyses have been conducted using flame ionization detectors (FID). These have the advantage of being sensitive and economical, but are nonspecific and provide no structural information.

Selective detectors confer distinct analytical advantages but are most often used to address special, nonroutine analytical problems. For example, the ability to detect a specific atom or molecular property can simplify sample preparation. Thus, by using a nitrogen/phosphorus-selective detector, contaminating compounds not containing nitrogen or phosphorus are simply not detected in most samples. Furthermore, it can reasonably be assumed that those peaks which have been detected contain nitrogen. In addition, problems caused by overlapping peaks are reduced. Selective detectors can also provide additional sensitivity. The ultimate detector is a mass spectrometer which can, depending on the context, provide all of these advantages and also provide detailed structural information.

A number of selective detectors have been used to assay amino acids. Their use will be illustrated using some examples.

A nitrogen/phosphorus-specific detector, operated in the nitrogen mode, has been used to assay free amino acids in conifer tissues. All the proteic amino acids and several biologically important nonproteic amino acids were assayed at the low picomole level as the N-HFB isobutyl esters. Comparison of the FID chromatogram with the NPD chromatogram enabled identification of those compounds which did not contain nitrogen (Figure 6). Similarly, 1-aminocyclo-propane-1-carboxylic acid, a precursor of the plant hormone ethylene, has been assayed as the N-benzoyl «-propyl derivative in the leaves and xylem sap of tomato plants. More recently (1997), 21 proteic and 33 nonproteic amino acids have been resolved in less than 30 min as the N-isobutoxycarbonyl methyl esters at a detection limit of 6-150 pg per injection. Small urine samples were analysed without prior clean-up and with no detectable influence from any non-nitrogen-containing compounds present.

Flame photometric detection (FPD) is useful for analysing sulphur-containing amino acids but has rarely been used in that context. Amino acid phos-phorylation is an important biochemical regulatory mechanism and is also important for correlating protein structure and function. The O-phosphoamino acids, specifically O-phospho serine, threonine and tyrosine, have been assayed as the N-isobutoxycar-bonyl methyl esters using FPD. The detection limits

Figure 6 Resolution of White Spruce leaf-free amino acids as the N-(O, S)-heptafluorobutyryl isobutyl esters using a flame ionization detector. Peaks marked by asterisks were shown not to contain nitrogen by comparison with an analysis of the same sample using a nitrogen-selective detector. (Reproduced with permission from MacKenzie SL (1986) Amino acid analysis by gas-liquid chromatography using a nitrogen-selective detector. Journal ofChromatography 358: 219-230.)

Figure 6 Resolution of White Spruce leaf-free amino acids as the N-(O, S)-heptafluorobutyryl isobutyl esters using a flame ionization detector. Peaks marked by asterisks were shown not to contain nitrogen by comparison with an analysis of the same sample using a nitrogen-selective detector. (Reproduced with permission from MacKenzie SL (1986) Amino acid analysis by gas-liquid chromatography using a nitrogen-selective detector. Journal ofChromatography 358: 219-230.)

ranged from 0.18 to 0.3 pmol, reflecting a sensitivity about 200 times greater than FID detection. The method has been applied to the determination of O-phosphoamino acids phosphorylated by protein kinase both in vitro and in vivo without radiolabell-ing. Other amino acids did not interfere. The secondary amino acids, proline, pipecolic, thioproline, hy-droxyproline and hydroxypipecolic acids, have also been assayed using FPD. Detection limits for the N-dimethylthiophosphoryl methyl esters were 0.1-0.7 pmol per injection.

Electron capture detectors are particularly useful for detection of the strongly electronegative per-fluoroacyl derivatives of amino acids, but few studies have been conducted. Typically, as little as 1.4 pmol of tyrosine has been detected in a standard amino acid mixture. y-Aminobutyric acid and five other aliphatic acids have been assayed in small volumes of super-natants from brain homogenates following sequential reaction with isobutyl chloroformate and penta-fluorophenol.

Mass spectrometric detection provides structural as well as quantitative information. It is most frequently used either to confirm the structure of derivatives during the development of new protocols or to identify unknown compounds. Detection limits are frequently in the femtomole range. Electron impact (EI) ionization is most commonly used but both positive and negative ion chemical ionization have also been applied. Selected ion monitoring (SIM) of diagnostic ions is often used to increase sensitivity.

Typical examples of the structural information role of a mass spectrometric detector are the identification of O-phosphoamino acids in urine hydrolysates, the identification of amino acid ethyl esters in wines, the determination of amino acid composition in small peptides, and the assaying of y-aminobutyric acid in mouse brain synaptosomes following therapy with the antiepileptic drug valproic acid. The versality of GC-mass spectrometry (GC-MS) is further illustrated by the identification of 3-OH-4-methyldecanoic acid, a fungal cyclodepsipeptide, and by the simultaneous analysis of branched-chain carboxylic, a-oxo, a-hy-droxy and a-amino acids in the urine of patients suffering from maple syrup urine disease. GC-MS has also been used to characterize binding media from medieval polychrome sculptures. Animal glues, casein, egg and drying oils were identified as components of the binders of paint and ground layers.

The expense of mass spectrometers mitigates against their use as routine analytical detectors and many real sample analyses by GC-MS (as distinct from the analysis of standard mixtures) have been directed to addressing analytical problems which cannot be resolved using other types of detectors.

The ratios of the stable isotopes of C and N are used in the assessment of in vivo protein turnover studies, and in identifying the sources and history of organic matter. Both natural abundances and the ratios obtained after enrichment with singly or multiply labelled amino acids or other compounds such as 13C-glucose, pyruvate or acetate have been determined. The ratios may be determined after online combustion following GC and introduction of the resultant gases into a conventional isotope ratio mass spectrometer. This approach has been used to study 15N: 14N isotopic ratios in plasma-free amino acids and, by eliminating many preparative steps, requires only about 500 ^L-of plasma, whereas preparatory methods may require as much as 60 mL.

Alternatively, the intact labelled compounds can be introduced directly into the mass spectrometer. For example, by combining stable isotope dilution with the use of EI and SIM to monitor the [M-57]# peak, homocysteine sulfinic acid, homocysteic acid, cystine sulfinic acid and cysteic acid have been shown to be agonistic to N-methyl-d-aspartate receptors in brain tissue. This approach also enabled the identification and quantitation of these compounds in normal human serum. Similarly, endogenous and newly synthesized concentrations of the neurotransmitter amino acids y-aminobutyric acid, glutamate and aspartate have been assayed in brain slices following incubation with 13C-labelled precursors.

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